Nuclear magnetic resonance (NMR) is a phenomenon exhibited by a select group of atomic nuclei and is based upon the existence of nuclear magnetic moments in these nuclei (termed "gyromagnetic" nuclei). When a gyromagnetic nucleus is placed in a strong, uniform and steady magnetic field (a so called "Zeeman field") an perturbed by means of a weak radio frequency (RF) magnetic field, it precesses at a natural resonance frequency known as a Larmor frequency, which is characteristic of each nuclear type and is dependent on the applied field strength in the location of the nucleus. Typical gyromagnetic nuclei include .sup.1 H (protons), .sup.13 C, .sup.19 F and .sup.31 P. The resonant frequencies of the nuclei can be observed by monitoring the transverse magnetization which results after a strong RF pulse. It is common practice to convert the measured signal to a frequency spectrum by means of Fourier transformation.
Although identical nuclei have the same frequency dependence upon the magnetic field, differences in the chemical environment of each nucleus can modify the applied magnetic field in the local vicinity of the nucleus, so that nuclei in the same sample do not experience the same net magnetic field. The differences in the local magnetic field result in spectral shifts in the Larmor frequencies between two such chemically non-equivalent nuclei, called "chemical shifts". These chemical shifts are interesting in that they reveal information regarding the number and placement of the atoms in a molecule and in the positioning of adjacent molecules with respect to each other in a compound.
Unfortunately, it is not always possible to interpret the frequency spectra produced by the chemical shifts because of other interfering and dominant interactions.
This is particularly true in NMR spectroscopy of solids. In liquid NMR spectroscopy, the rapid motion of the liquid molecules tends to isolate the nuclei and separate the nuclear interactions, so that it is easier to distinguish separate nuclei in the final output. In solid state NMR, there are many interactions between the molecules which obscure the output. For example, the magnetic moments in neighboring nuclei perturb each other, resulting in interactions called dipole-dipole couplings. These couplings tend to broaden the characteristic resonance peaks and obscure the "fine" resonant structure produced by the chemical shifts. An additional problem found in solids, which is not present in liquids, is that the orientation of the solid molecules is relatively fixed with respect to the applied Zeeman field and, accordingly, the chemical shifts are anisotropic, in that a component of the resonant frequency depends on the physical orientation of the molecules with respect to the applied field.
Therefore, it is essential to suppress some interactions over others to obtain a meaningful output. This is usually done by perturbing the system at selected frequencies to cause unwanted interactions to cancel or average to a reduced amplitude. For example, in solids, the aforementioned chemical shift anisotropy is usually greatly reduced by orienting the solid sample at a "magic angle" (54'44") with respect to the applied Zeeman field and physically rotating the solid at a relatively rapid rate causing the anisotropic field components to average to zero.
Similarly, by well-known techniques, it is possible to reduce the unwanted spin-spin interactions by irradiating the nuclei with additional pulses of RF energy at or near the Larmor frequencies. By properly selecting various orientations and phases of the RF pulses, the polarization of the perturbing nuclear spin systems in neighboring groups can be changed, effectively averaging out the spin interactions so that the contribution to the final output is greatly diminished. Since the Larmor frequencies for each nuclear type are distinct, an applied RF frequency will have a much greater effect on those nuclei which have a Larmor frequency which is close to the applied frequency than those nuclei in which the Larmor frequency is considerably different. Thus, the applied RF fields can be used to affect one type of nucleus while leaving others unchanged.
Due to the special problems encountered in solids, it is common to use a two-dimensional time domain spectroscopic technique to obtain increased resolution. With this technique it is possible to study the interaction or "correlation" between two different types of gyromagnetic nuclei in a solid--the interaction between protons and .sup.13 C nuclei is typically of interest in many organic solids. The basic techniques of two-dimensional heteronuclear correlation as applied to solids are well-known and described in many articles, such as "Heteronuclear Solid State Correlation Spectroscopy", P. Caravatti, G. Bodenhausen and R. R. Ernst, Chemical Physics Letters, Vol. 89, No. 5, pp. 363-367 (July 1982) and "Heteronuclear Correlation Spectroscopy In Rotating Solids", P. Caravatti, L. Braunschweiler and R. R. Ernst, Chemical Physics Letters, Vol. 100, No. 4, pp 305-310 (September 1983) which articles are hereby incorporated by reference.
As described in the aforementioned articles, the basic two-dimensional heteronuclear correlation technique involves performing a process or "experiment" in the time domain, consisting of four distinct, sequential time intervals. The first time period is called is a "preparation" period. During this period, one of the two nuclei types to be studied is placed in a excited, coherent non-equilibrium state, which state will change or "evolve" in the subsequent time periods. The preparation time period may consist of the application of a single RF pulse to the system, or a sequence of RF pulses. The preparation period normally has a fixed time duration.
The second time period is called an "evolution" period during the course of which the excited nuclei evolve under the influence of the applied magnetic field, neighboring nuclei, any applied periodic RF pulse sequences and physical sample spinning. The evolution of the excited nuclei during this period allows these frequencies to be determined. A series of "experiments" or "scans" are carried out with a systematic incrementation of the evolution time period between each experiment.
The evolution period is followed by a "mixing" period. During the mixing period one or more RF pulses may be applied, which cause the transfer of coherence or polarization from the excited nuclei to the other nuclear type under observation. The transfer of coherence or polarization induced by the mixing process is characteristic of the nuclear system under investigation.
The mixing period is followed by a "detection" period in which the resonance frequencies of the second nuclear type are is measured. During this period it is conventional to apply further pulses or continuous wave RF energy to prevent further interaction of the two nuclear types.
After Fourier transformation, the result of the multiple experiments is a two-dimensional spectral "plot" called a heteronuclear correlation spectrum (also called a 2D HETCOR plot). One axis of this plot is the detected frequencies of the second nuclear species. The other axis represents the frequencies of the first nuclear species as determined from repeated scans or experiments with incremented evolution times. Since the output frequencies of the second nuclear species are dependent on the transfer of energy from the initially-excited first nuclear species and the state of the first nuclear species depends, in turn, on the evolution time, the second plot axis effectively represents the chemical shifts due to the various first nuclear species in a particular molecule and their spatial relation to the second species. Thus output peaks on the plot represent correlations between the first and second nuclear species of selected nuclei in a given molecule. One advantage of heteronuclear correlation is that it separates the proton resonance over the much larger .sup.13 C chemical shift range. Therefore, the technique can provide well resolved proton chemical shift information for samples where it is impossible to resolve the proton chemical shifts with any standard, one-dimensional spectroscopic technique.
For example, in a typical two-dimensional heteronuclear correlation experiment applied to an organic material, it is common to study the correlation between the hydrogen, .sup.1 H nuclei (protons), and .sup.13 C nuclei within the sample. In order to do this, during the preparation period, an RF pulse is applied which excites the hydrogen protons. In theory, the protons would then freely evolve during the evolution period. During the mixing period, the protons interact with the .sup.13 C nuclei through direct heteronuclear dipole-dipole coupling. Finally, during the detection period, the .sup.13 C frequencies are detected. One of the advantages of such an experiment is that the heteronuclear coupling between the protons and .sup.13 C nuclei depends entirely on the distance between the nuclei, regardless of chemical bonding. Thus, the correlation provides a method for studying the stereochemistry of individual molecules and the positioning of adjacent molecules relative to each other.
The problem with this technique is that other couplings, such as a "homonuclear" dipole-dipole coupling between protons and the "heteronuclear" dipole-dipole coupling between protons and carbon nuclei can obscure the desired output if these interactions are allowed to occur during the evolution time period because they affect the measurement of the proton chemical shifts. These latter two interactions cause spreading of the proton chemical shift peaks which results of overlap of separate proton sites, in turn, obscuring the various separate sites. Thus, it is necessary to suppress these two very strong interactions during the evolution time period. In certain circumstances, where a more abundant element is being observed instead of .sup.13 C, for example, phosphorous or aluminum, it may also be necessary to suppress the homonuclear interaction between these latter nuclei.
In general, in order to accomplish the suppression of homonuclear and heteronuclear couplings during the evolution period, carefully designed pulse sequences of RF pulses are applied to either the protons, the .sup.13 C nuclei or both simultaneously during the evolution period. The purpose of these pulse sequences is to suppress or average the results of the unwanted interactions. Many RF pulse sequences of this type are well known in the prior art.
For example, in the prior art, RF pulse sequences were known which were relatively efficient for suppressing homonuclear interactions between the protons. In addition, other pulse sequences were known for suppressing heteronuclear interactions between the protons and the .sup.13 C nuclei. In an attempt to simultaneously suppress both homonuclear and heteronuclear interactions, the prior art simply combined the known RF pulse sequences. Since the existing RF pulse sequences were never intended to be used in combination, the result was that a very long sequence of RF pulses was required to suppress both types of interactions, and the method did not work very well. Consequently, the number of inequivalent proton sites that could be resolved was severely limited, in turn, limiting the number of possible compounds that could be effectively studied.
Accordingly, it is an object of the present invention to improve resolution of a conventional two-dimensional NMR correlation experiment.
It is another object of the present invention to increase the number of proton sites which can be resolved in a conventional two-dimensional NMR correlation experiment.
It is still another object of the present invention to provide a new RF pulse sequence which improves resolution of a conventional two-dimensional NMR correlation experiment.
It is yet another object of the present invention to provide a new RF pulse sequence for use during the evolution period of a two-dimensional NMR correlation experiment, which pulse sequence can effectively suppress heteronuclear dipolar coupling.
It is a further object of the present invention to provide new RF pulse sequences, which can be used in conjunction with other pulse sequences during the evolution period of a two-dimensional NMR correlation experiment to effectively suppress homonuclear and heteronuclear interactions.